Development of two age estimation models for buccal swab samples based on 3 CpG sites analyzed with pyrosequencing and minisequencing

May 2, 2021Forensic science international. Genetics

Two models to estimate age from cheek swab DNA using three specific DNA markers

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Abstract

A mean absolute deviation of 5.33 years was observed when estimating age using DNA methylation analysis of buccal swabs.

Age-dependent methylation was analyzed at 88 specific CpG sites across eight marker regions.
141 buccal swab samples from individuals aged 21 to 69 years were used for the study, split into training and validation sets.
Three CpG sites were identified that correlated well with age, leading to an age estimation model based on these markers.
The pyrosequencing model showed a mean absolute deviation of 5.33 years, while the minisequencing model had a mean absolute deviation of 6.44 years.
Both methods demonstrated high concordance in results, indicating reliability in age estimation.
Buccal swabs provide a non-invasive alternative to blood samples for molecular age estimation.

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The analysis of DNA methylation levels of specific CpG sites is one of the most promising molecular techniques to estimate an individual's age. Numerous studies were published recently presenting age estimation models based on DNA methylation patterns from blood samples, with only a few using saliva or buccal swabs. The aim of this study was to identify age-dependent methylation of 88 CpG sites in eight different marker regions (PDE4C, ELOVL2, ITGA2B, ASPA, EDARADD, SST, KLF14 and SLC12A5) in buccal swab samples. A total of 141 buccal swabs from individuals with age ranging from 21 to 69 years were split into a training set (n = 95) and a validation set (n = 46). Samples of the training set were analyzed by pyrosequencing and markers with best age correlation were identified. Stepwise linear regression analysis was performed resulting in an age estimation model including three of the examined CpG sites and showing a mean absolute deviation of estimated from chronological age of 5.11 years. To allow easy implementation into forensic laboratories without the need for pyrosequencing equipment, a multiplex minisequencing reaction was developed, including the same CpG sites previously identified by pyrosequencing. An adjusted age estimation model was evaluated with a mean absolute deviation of estimated from chronological age of 5.16 years. The independent validation set of 46 buccal swab samples was used to test model performances. Mean absolute deviation of estimated from chronological age was 5.33 years and 6.44 years for the pyrosequencing model and the minisequencing model, respectively. Comparison of the two methods showed a high concordance of results, both, qualitatively and quantitatively. In conclusion, buccal swabs offer a suitable alternative to blood samples for molecular age estimation with the additional advantage of being collected non-invasively. Furthermore we showed that minisequencing offers a cost-effective and easy-to-integrate alternative to pyrosequencing for the analysis of methylation status of individual CpG sites.

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Development of two age estimation models for buccal swab samples based on 3 CpG sites analyzed with pyrosequencing and minisequencing

Abstract

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